1. Field of the Invention
This invention relates generally to methods for increasing the yield of a compound produced by an organism. More particularly, the present invention relates to methods for increasing the total or soluble carbohydrate content or sweetness or increasing the content of an endogenous carbohydrate of a plant tissue by producing a sugar-metabolizing enzyme that catalyzes the conversion of an endogenous sugar (one that is normally produced in the plant) to an alien sugar (one that is not normally produced in the plant at the same developmental stage). The invention also relates to plants and plant parts that produce a sugar-metabolizing enzyme to yield an alien sugar, with the consequence of higher total fermentable carbohydrate content, and to fermentable carbohydrates and other products derived therefrom.
Bibliographic details of the publications referred to in this specification are collected at the end of the description.
2. Description of the Related Art
Plants are the primary source of renewable bioenergy, biomaterials and feedstocks for industrial biotransformations. The yield and concentration of desired sugars in plants are key determinants of the technical and economic feasibility of downstream industrial processes. However, the metabolic networks of plants for biosynthesis of sugars show substantial internal buffering and redundancy, with the consequence that alteration to a key gene in metabolism of a sugar commonly results in no useful change to the harvestable yield of the sugar (Moore, 1995; Nguyen-Quoc and Foyer, 2001; Fernie et al., 2002). For example, Botha et al. (2001) have shown that 70% reduction in activity of a key enzyme in sucrose breakdown (acid invertase) results in no significant change in sucrose yield or purity in transgenic sugarcane.
Sucrose isomerases are enzymes produced by organisms including various microbes, with the capability to convert the disaccharide sucrose into isomers such as isomaltulose (palatinose) or trehalulose. Sucrose isomerases vary in their properties including the disaccharide reaction products, the proportion of monosaccharides such as glucose and fructose in the reaction products, the kinetic properties of the enzymes, the optimal reaction conditions, and the sensitivity of the enzyme to variations from the optimal conditions (Veronese and Perlot, 1999).
Sucrose is the major intermediary in carbon flux between source (photosynthetic) tissues and sink (growth and storage) tissues within plants, and it is the primary storage product in certain plants such as sugarcane and sugarbeet. Several reports indicate that expression of introduced sucrose isomerase genes in plants can result in the conversion of sucrose to an isomer such as isomaltulose, and it has been envisaged that this conversion could be beneficial for the industrial production of the isomer. The emphasis has been towards the high-level or complete conversion of sucrose to the isomer as a desired industrial product (Birch and Wu, 2002; Börnke et al., 2002b), or as a precursor for in planta conversion to derived industrial materials (Kunz et al., 2002).
It has also been envisaged that the conversion could be lethal to plant cells by conversion of an essential carbon and energy reserve to an unavailable form, with applications in cell ablation for purposes such as engineered male sterility (Bornke and Sonnewald, 2001). Indeed, several reports indicate that sucrose isomerase transgene expression is harmful to plant development and yield, causing severe growth abnormalities, reduced starch content, and reduced soluble carbohydrate content (Börnke et al., 2002a, b).
In tobacco, there were severe and damaging effects on plant development due to constitutive expression from the CaMV35S promoter of a sucrose isomerase gene fused to the potato proteinase inhibitor II signal peptide to direct the sucrose isomerase enzyme to the extracellular (apoplasmic) space. Low isomaltulose levels (0.3 to 0.6 mM m−2 of leaf tissue) were detected, equivalent to about 20% to 44% of the normal carbohydrate levels in leaf starch or 10 to 45 times the normal low transitory sucrose levels in this source tissue. Effects on stored carbohydrates in sink tissues were not reported. Growth of leaves and other organs was severely disrupted, and the plants were unable to reproduce (Börnke et al., 2002a).
In potato, expression of the same apoplasm-targeted sucrose isomerase from the tuber-specific patatin B33 promoter resulted in plants without apparent adverse effects on growth and development. Isomaltulose yields were again low (10-15 μmol g−1 fresh weight of tuber tissue, equivalent to about 4% to 5% of the normal stored carbohydrate levels in tuber starch), and slightly below the usual sucrose levels in this starch-storing sink tissue. Furthermore, because of accompanying decreases in contents of sucrose, hexoses and starch, yields of total soluble carbohydrate (excluding starch) and total fermentable carbohydrate (including starch) were decreased in the modified lines (Börnke et al., 2002b).
To overcome these problems, the present inventors conceived a novel approach based upon combining (i) a sucrose isomerase enzyme, especially a highly efficient sucrose isomerase such as UQ68J; (ii) use of a plant species such as sugarcane that accumulates sucrose as the stored carbohydrate; and (iii) targeting the introduced sucrose isomerase to the sucrose storage compartment, for example the large vacuole of sucrose storage parenchyma within the mature culm in the case of sugarcane. Because isomaltulose is not metabolized in plants, the present inventors hypothesized that, in contrast to sucrose, it might not be subject to ‘futile cycles’ of degradation and synthesis in the mature storage tissues, which have the potential to decrease storage efficiency and harvestable yield. Therefore, the inventors' approach was designed to achieve higher yields of soluble carbohydrates and fermentable carbohydrates in the modified plants, in contrast with the reduction of these yields reported from previous approaches. Consistent with this hypothesis, it was found that isomaltulose concentrations above 500 mM in juice can be achieved in sugarcane, by expressing a sucrose isomerase (e.g., the highly efficient sucrose isomerase UQ68J), targeted to the sucrose storage vacuoles in mature stem parenchyma. This exceeds the total stored carbohydrate content obtained from unmodified sugarcane, and it may be accomplished without commensurable reduction in the content of endogenous sugars, resulting in a much higher total soluble sugar content in the modified lines.
Plants have highly adapted sensors and transporters for sucrose, but it is generally considered that these sucrose sensors and transporters are not able to respond in the same way to isomers such as isomaltulose (Loreti et al., 2000; Sinha et al., 2002). In stark contrast with sucrose, plants are unable to metabolize some isomers such as isomaltulose as a source of carbon and energy (Sinha et al., 2002). Nevertheless, the isomers can elicit changes in the patterns of cellular gene expression and modify the activities of certain enzymes involved in sucrose metabolism or in signal-transduction cascades in plants (Fernie et al., 2001; Sinha et al., 2002).
Because exogenous supply of isomaltulose to tissue slices from potato tubers altered the metabolism of other exogenously supplied sugars, Fernie et al. (2001) suggested that supplying isomaltulose to potato tubers represents a novel way to increase starch synthesis. However, the exogenous supply of a substance like isomaltulose to plant organs such as potato tubers is unlikely to be practical for industrial use, and there is no report that this approach has been tested or applied to enhance starch yield. In studies by Börnke et al. (2002b), transgenic potato plants expressing an apoplasmic sucrose isomerase gene from a tuber-specific promoter accumulated isomaltulose to a level approaching the usual sucrose content in tubers, but showed decreased yield of starch and of total soluble sugars.
Based on consideration of the differential capacity of plants to sense sucrose versus related compounds such as isomaltulose, the present inventors conceived another approach to achieve increased yields of endogenous sugars in plants by appropriate expression of an introduced sucrose isomerase. This contrasts with the intent of previously explored strategies (to produce plants for the harvest of isomaltulose or derivatives of isomaltulose), and with their outcome (plants with reduced yields of endogenous carbohydrates). Because the signaling and control mechanisms that operate on plant metabolism are incompletely understood, the present inventors undertook substantial experimentation to determine a scope of conditions yielding their desired industrial outcome (plants with increased yields of endogenous carbohydrates). In this regard, it was found that total soluble sugar contents in the range of 700-900 mM sucrose equivalents in juice can be achieved in sugarcane lines engineered for low-level expression of a sucrose isomerase directed to the cytosolic compartment, or divided between compartments. This is approximately twice the total stored carbohydrate content typically obtained from unmodified sugarcane, and it may be accomplished with little change to the harvested sugar composition. The approach is not limited to the sucrose isomerase gene, the isomaltulose conversion product, or the sugarcane plant used by way of example. It encompasses more broadly the expression within an organism of an introduced gene resulting in the partial conversion of a substrate endogenous compound that is normally sensed by the organism into a product compound that is not perceived in an equivalent manner within the organism, with the effect that metabolic flows are altered, resulting in the accumulation of higher yields of desired endogenous compounds.